Alfalfa (Medicago sativa) is the most cultivated legume worldwide, with the US being the top alfalfa producer. The methods of biotechnology have been applied to alfalfa for improvement of agronomic traits and the quality of the product. One such agronomic trait is lignin content.
Lignin is the second most abundant terrestrial biopolymer and accounts for 30% of the organic carbon. Lignin is crucial for structural integrity of the cell wall and it imparts stiffness and strength to the stem. Lignin content is inversely correlated with forage digestibility for diary cattle. A reduction in lignin may be achieved in transgenic plants by the expression of a RNA suppression construct capable of providing such decrease while at the same time provide increased alfalfa digestibility. The expression of foreign genes or suppression molecules in plants is known to be influenced by many factors, such as the regulatory elements used, the chromosomal location of the transgene insert, the proximity of any endogenous regulatory elements close to the transgene insertion site, and environmental factors such as light and temperature. For example, it has been observed that there may be variation in the overall level of transgene suppression or in the spatial or temporal pattern of transgene suppression between similarly-produced events. For this reason, it is often necessary to screen hundreds of independent transformation events in order to ultimately identify one event useful for commercial agricultural purposes. Such an event, once identified as having the desired suppression phenotype, molecular characteristics and the improved trait, may then be used for introgressing the improved trait into other genetic backgrounds using plant breeding methods. The resulting progeny would contain the transgenic event and would therefore have the same characteristics for that trait of the original transformant. This may be used to produce a number of different crop varieties that comprise the improved trait and are suitably adapted to specific local growing conditions.
It would be advantageous to be able to detect the presence of transgene/genomic DNA of a particular plant in order to determine whether progeny of a sexual cross contain the transgene/genomic DNA of interest. In addition, a method for detecting a particular plant would be helpful when complying with regulations requiring the pre-market approval and labeling of foods derived from the transgenic crop plants.
The presence or absence of a suppression element may be detected by any well known nucleic acid detection method such as the polymerase chain reaction (PCR) or DNA hybridization using nucleic acid probes. These detection methods generally focus on frequently used genetic elements, such as promoters, terminators, marker genes, etc. As a result, such methods may not be useful for discriminating between different transformation events, particularly those produced using the same DNA construct unless the sequence of chromosomal DNA adjacent to the inserted DNA (“flanking DNA”) is known. An event-specific PCR assay is discussed, for example, by Taverniers et al. (J. Agric. Food Chem., 53: 3041-3052, 2005) in which an event-specific tracing system for transgenic maize lines Bt11, Bt176, and GA21 and for canola event GT73 was demonstrated. In this study, event-specific primers and probes were designed based upon the sequences of the genome/transgene junctions for each event. Transgenic plant event specific DNA detection methods have also been described in U.S. Pat. Nos. 7,632,985; 7,566,817; 7,368,241; 7,306,909; 7,718,373; 7,189,514, 7,807,357 and 7,820,392.